Kluyveromyces marxianus, a non-conventional yeast, carries traits deemed suitable for industrial applications, such as ethanol production, exhibiting advantages over Saccharomyces cerevisiae in terms of growth rate and thermotolerance. Non-invasive parallel monitoring of biomass in shake flask cultures allows for efficient microorganism characterization, providing much-needed and accurate data on these strains. Therefore, this study aimed to assess the behavior of two K. marxianus strains during continuous shake flask cultivation. Strains IZ 1339 (native strain isolated from a drosophilid) and FT 146L (isolated from ethanol production) were utilized in this study. The cultivations were performed utilizing sterile sugarcane molasses, diluted to 8 and 15 ºBrix (M8 and M15, respectively). Yeast strains were pre-grown on YPD 2% media, adjusted to OD600 1.6 and inoculated (1 mL aliquot) on 50 mL of molasses (M8 and M15) in 250 mL flasks, in duplicates. Biomass was monitored online and non-invasively by Cell Growth Quantifier (Aquila Biolabs). Cultivations were performed at 30 °C for 24 hours. Strain IZ 1339 presented a continuous growth curve during the cultivation period, reaching ODmax at 24 h, for both M8 (13.23) and M15 (13.19). Maximum growth rate for IZ 1339 was 0.25 h-1 at 6 h for M8 and 0.18 h-1 at 9 h for M15. For strain FT 146L, ODmax was reached at 15 h for M8 (9.64), and 24 h for M15 (7.91). Maximum growth rate for FT 146L was 0.19 h-1 at 8 h for M8 and 0.20 h-1 at 10 h for M15. Strain IZ 1339 exhibited a constant, however, slower growth pattern when compared to strain FT 146L, which grew constantly up until the 12 h mark, after that the strain presented flocculation, impacting the quality of the readings. Strain IZ 1339 also had a higher ODmax value when compared to FT 146L, nevertheless, their growth rate was similar, showing that both strains had a satisfactory performance in both concentrations of molasses. Considering that ethanol production in Brazil performs fermentation in a must of around 20 ºBrix, it is interesting how both strains tolerated and also performed successfully in both concentrations, considering their role as potential ethanol-producing strains. Non-invasive monitoring makes it possible to accompany the growth pattern of the strains, indicating that both K. marxianus strains perform well when grown in a sugarcane molasses medium. This feature makes these K. marxianus strains an interesting non-conventional alternative to S. cerevisiae when it comes to industrial application.